Neurexin–neuroligin signaling in synapse development
Ann Marie Craig and Yunhee Kang
Neurexins and neuroligins are emerging as central organizing
molecules for excitatory glutamatergic and inhibitory
GABAergic synapses in mammalian brain. They function as cell
adhesion molecules, bridging the synaptic cleft. Remarkably,
each partner can trigger formation of a hemisynapse:
neuroligins trigger presynaptic differentiation and neurexins
trigger postsynaptic differentiation. Recent protein interaction
assays and cell culture studies indicate a selectivity of function
conferred by alternative splicing in both partners. An insert at
site 4 of b-neurexins selectively promotes GABAergic synaptic
function, whereas an insert at site B of neuroligin 1 selectively
promotes glutamatergic synaptic function. Initial knockdown
and knockout studies indicate that neurexins and neuroligins
have an essential role in synaptic transmission, particularly at
GABAergic synapses, but further studies are needed to assess
the in vivo functions of these complex protein families.
Addresses
Brain Research Centre, University of British Columbia, 2211 Wesbrook
Mall, Vancouver V6T 2B5, Canada
Corresponding author: Craig, Ann Marie (amcraig@interchange.ubc.ca)
Current Opinion in Neurobiology 2007, 17:43–52
This review comes from a themed issue on
Development
Edited by Ben Barres and Mu-Ming Poo
0959-4388/$ – see front matter
# 2006 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.conb.2007.01.011
Introduction
Neurexin 1a was first identified in 1992 [1] by affinity
chromatography of rat brain extract on a column of
a-latrotoxin (a component of black widow spider venom
that stimulates massive synaptic vesicle fusion). Over the
subsequent 15 years, pioneering studies by Sudhof and
colleagues have characterized neurexins and their binding partners the neuroligins. The intense current interest
in these proteins was triggered by the finding of Scheiffele et al. [2] that neuroligins presented on the surface of
non-neuronal cells induce synaptic vesicle clustering and
formation of functional release sites in contacting glutamatergic axons. A complementary study by Graf et al. [3]
then showed that neurexins presented alone to dendrites
trigger postsynaptic differentiation, inducing clusters of
GABA postsynaptic components even more so than
glutamate postsynaptic components. Overexpression
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and knockdown of neuroligins in culture led to the idea
that these molecules control the balance of GABAergic
and glutamatergic inputs [4,5]. We focus our discussion
here on studies from the past two years that address how
alternative splicing in both neurexin and neuroligin transcripts regulates their function. We also discuss initial
results from studies of knockout mice and disease-linked
mutations.
Structure of neurexins and neuroligins
There are three neurexin genes in mammals, each of
which has both an upstream promoter that is used to
generate the larger a-neurexins and a downstream promoter that is used to generate the b-neurexins (Figure 1)
[6]. Alternative splicing at five sites and N- and O-glycosylation contribute additional diversity. By contrast,
Drosophila melanogaster and Caenorhabditis elegans have
only a single a-neurexin homolog. b-Neurexins contain
a single LNS domain (laminin, neurexin, sex-hormonebinding protein domain; also known as a Laminin G
domain), whereas a-neurexins contain six LNS domains
organized into modules with three EGF-like domains.
The crystal structure of the neurexin 1b LNS domain
revealed a remarkable conservation with the agrin LNS
domain in the position of alternative splice sites [7]. This
structural similarity might reflect functional similarity.
Alternative splicing of agrin regulates its role as an
essential synaptic organizer at the mammalian neuromuscular junction [8]; as discussed later in this review,
alternative splicing of neurexin regulates its role at
synapses in the CNS. A recent structure–function study
has confirmed that binding to neuroligins and synaptogenic activity is mediated by same face of the neurexin
1b LNS domain that contains the splice sites (Figure 1)
[9]. The crystal structure of the second LNS domain of
neurexin 1a revealed that this region containing the
splice sites forms a highly variable surface surrounding
a coordinated Ca2+ ion [10]. Ca2+ binding occurred with
low affinity (Kd 400 mM) and was reduced to below
detectable levels by addition of the eight-residue or
fifteen-residue splice inserts. Thus, some of the Ca2+dependent interactions of neurexins might be influenced
by reductions in cleft Ca2+ concentrations following
synaptic activity (but note that interaction of neurexin
1b with neuroligin 1 was half-maximal at Ca2+ concentrations of 2 mM [11], which is well below the estimated
range in the synaptic cleft).
Neurexins have three known extracellular binding partners in the mammalian brain: neuroligins, dystroglycan
and neurexophilins. Neuroligins exhibit Ca2+-dependent
binding to a-neurexins and b-neurexins, dystroglycan
Current Opinion in Neurobiology 2007, 17:43–52
44 Development
Figure 1
Structure of neurexins and neuroligins. In humans, there are three neurexin genes and five neuroligin genes. Each neurexin gene uses an
upstream promoter to generate the larger a-neurexins and a downstream promoter to generate the smaller b-neurexins. Thus, b-neurexins
can be thought of as N-terminally truncated a-neurexins that have a short b-specific leader (bN). In a-neurexins, the LNS (laminin, neurexin,
sex-hormone-binding protein) domains are organized with EGF (epidermal growth-factor)-like domains into three homologous modules, I–III.
The position of each of five sites of alternative splicing (SS1–SS5) is indicated. Neuroligins contain an extracellular acetylcholinesterase
(AChE)-homologous domain that contains one or two sites of alternative splicing (SSA, plus SSB in the case of neuroligin 1). Both neurexins
and neuroligins contain a highly glycosylated region (CH) and a transmembrane domain (TM; not present in some splice variants of neurexin 3),
and terminate in PDZ-domain-binding sites (PDZ BD). Shown between the neurexins and neuroligins are structures of AChE, a model for
the AChE-homologous domain of neuroligins, and the neurexin 1b LNS domain [7]. The position of splice sites SS2–SS4 is shown on a
single LNS domain for simplicity, although SS2 and SS3 actually occur in different LNS domains of a-neurexins. Note also that the left face
of the neurexin LNS as shown here binds neuroligin [9] but the precise structure and interacting region of neuroligin has not been reported
yet. The structure files E.C.3.1.1.7 (AChE) and d1c4ra (neurexin LNS) were downloaded from the Research Collaboratory for Structural
Bioinformatics protein data bank (http://www.rcsb.org/pdb/home/home.do) and visualized using the program Visual Molecular Dynamics [66].
shows Ca2+-dependent binding preferentially to a-neurexins, and neurexophilin binding is Ca2+-independent
and specific to a-neurexin. Neuroligins were first identified using an affinity column of neurexin 1b and they are
the most intensively studied neurexin binding partners.
There are five neuroligin genes in humans: NLGN1,
NLGN2, NLGN3, NLGN4 and NLGN4Y [12]. The major
extracellular domain of neuroligins is homologous to
acetylcholinesterase (AChE) but lacks cholinesterase
activity and mediates binding to neurexins. Curiously,
overexpression of AChE reduces levels of b-neurexins
in vivo and in culture, and impairs genesis of glutamatergic synapses in culture, indicating that there is crosstalk between the two proteins [13,14]. Neuroligins form
Current Opinion in Neurobiology 2007, 17:43–52
homo-multimers through the AChE-homologous domain,
a structural association that is important for neuroligin
function [15,16]. However, hetero-oligomerization versus
homo-oligomerization is a potential regulatory step that
has not yet been explored. The AChE-homologous region
of neuroligins contains alternative splice site A, and there
is an additional splice site B within this region specifically
in neuroligin 1 (Figure 1). Thus, both neuroligins and
neurexins exist in numerous isoforms that derive from
multiple genes and alternative splicing. Neuroligins and
neurexins both have relatively short intracellular domains
that terminate in PDZ-domain-binding sites, which are
presumably important for linking them to other synaptic
proteins.
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Neurexin–neuroligin signaling in synapse development Craig and Kang 45
Synaptic localization and interacting partners
Because neurexin 1a can function as an a-latrotoxin
receptor to mediate transmitter release [17], it is assumed
that neurexins at least partially localize to the presynaptic
terminus; this assumption has been confirmed by antibody labeling and localization of tagged recombinant
neurexins [1,3,16]. Ultrastructural localization using
high-quality antibodies is still needed to define the precise localization of neurexins in relation to different
synapse types during development. At the cellular level,
the six major neurexin forms (1a, 2a, 3a, 1b, 2b and 3b)
show fairly broad overlapping expression patterns in
brain, such that most neurons express multiple neurexins
[18]. Thus, there is not an obvious segregation according
to transmitter type.
Extracellularly, the functional significance of neurexins
binding to neuroligins is becoming appreciated, as we will
go on to discuss extensively, but the significance of the
interaction of neurexins with dystroglycan or neurexophilins is not so clear. Dystroglycan binds either to the
LNS domain that is common to both a-neurexins and bneurexins or to the second LNS domain of a-neurexins,
and in both cases binds only to LNS domains lacking
splice inserts [19]. Mice that have brain-specific deletion
of dystroglycan exhibit impaired long-term potentiation
and developmental malformations characteristic of muscular dystrophy, but they have relatively normal baseline
synaptic transmission [20]. Given the selective concentration of dystroglycan to a subset of mature GABAergic
synapses [21,22], it might be informative to study in detail
GABA-mediated transmission in mice lacking neuronal
dystroglycan, both in the single-mutant mice and in
knockouts that also lack neuroligins.
Neurexophilins are secreted peptides that are processed
proteolytically and that bind to the second LNS domain
of a-neurexin. The two neurexophilins that bind a-neurexins in mice, Nxph1 and Nxph3 — NXPH2 is expressed
only in humans — show very restricted expression patterns [23,24]. Each single-knockout mouse shows normal
brain morphology, and a Nxph1;Nxph3 double-knockout
mouse is viable. However, the increased startle responses
and impaired motor coordination of the Nxph3 knockout
mice indicate that neurexophilins have a functional role
in specific circuits [24].
Intracellularly, the C termini of neurexins bind to synaptotagmin and to the PDZ domains of CASK, syntenin and
Mint [25–28]. These interactions constitute a link from
neurexins to both synaptic vesicles and the vesicle fusion
apparatus.
Neuroligins bind to several postsynaptic components of
glutamatergic synapses: PDZ-domain scaffolding
proteins such as PSD-95 and related MAGUKs, S-SCAM
and related MAGIs, and probably also Shank, PICK1,
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GOPC and SPAR [29–31]. Thus, it came as a surprise when
two groups [3,32] independently found that neuroligin 2
concentrates not at glutamate postsynaptic sites but
at GABA postsynaptic sites. This is in contrast to the
glutamatergic postsynaptic localization of neuroligin 1
(Figure 2) [33]. The mechanisms by which neuroligin 2
is inhibited from binding to glutamate-specific PDZ
domain proteins such as PSD-95 in neurons, how it localizes to GABAergic synapses and which proteins it binds to
in neurons are questions under active investigation.
A dendritic-targeting motif identified in the central portion
of the neuroligin 1 cytoplasmic domain seems to be
conserved among the neuroligins and distinct from the
synaptic-targeting regions [34].
Triggers of presynaptic and postsynaptic
differentiation
Neuroligins presented alone on the surface of nonneuronal cells or beads induce localized formation of
functional release sites in axons, by aggregating neurexins
on the axon surface [2,16]. Neuroligins induce formation
of glutamatergic and GABAergic presynaptic specializations, with neuroligin 2 being relatively more active on
GABAergic axons [3,5,35]. The neuroligin-induced presynaptic specializations exhibit release characteristics
remarkably similar to those of bona fide synapses and
might be considered as hemisynapses. A striking demonstration of the function of these hemisynapses is the
miniature excitatory postsynaptic current (mEPSC)-like
events that occur in HEK cells expressing neuroligins and
glutamate receptors and co-cultured with neurons [36,37].
Neurexins, like their neuroligin binding partners, also
signal trans-synaptically. Neurexins presented alone on
the surface of non-neuronal cells or beads induce localized clustering of neurotransmitter receptors and other
postsynaptic components, by aggregating neuroligins on
the dendrite surface [3,38]. Neurexins induce clustering
of several scaffolding and signaling proteins that are
characteristic of glutamatergic and GABAergic synapses,
with a selective link from neuroligin 2 to components of
GABAergic synapses [3]. An important exception is
AMPA receptors: these were not aggregated by neurexins
alone but seem to require additional signals, including
stimulation of the aggregated NMDA receptors and
perhaps increased levels of PSD-95 [38].
Several studies have assessed the function of neuroligins
by overexpression of full-length or truncated versions in
neurons; these increase or decrease synaptic protein
accumulation, mEPSCs and miniature inhibitory postsynaptic currents (mIPSCs), depending on the construct and
expression level (reviewed recently in [39]). A particularly
interesting finding of these and related studies is that the
level of the interacting protein PSD-95 can influence the
level of neuroligins at excitatory versus inhibitory
synapses, and the balance of functional excitatory versus
Current Opinion in Neurobiology 2007, 17:43–52
46 Development
Figure 2
Molecular interactions at glutamatergic and GABAergic synapses that are linked by neurexins and neuroligins. (a) A glutamatergic synapse.
(b) A GABAergic synapse. The broken lines between neuroligin 2 and GABA receptors (GABAR) and Gephyrin indicate that there are some links
(direct or indirect) but their nature is not yet known. At glutamatergic synapses, a-neurexins can bind neuroligin 1 that lacks an insert at the B
splice site ( B) but the majority of neuroligin 1 is in the +B form, which does not bind to a-neurexins. Additional abbreviations: AMPAR,
AMPA receptor; NMDAR, NMDA receptors; VGAT, vesicular GABA transporter; VGlut1, vesicular glutamate transporter.
inhibitory input [4,35,40]. For example, overexpression of
PSD-95 can redirect neuroligin 2 from excitatory to inhibitory synapses, reducing inhibitory input while strengthening excitatory synapses. The levels of neuroligins 1, 2 and 3
have been reduced using RNA interference (RNAi) in
hippocampal cultures [5]. Knockdown of all three neuroligins, and to a lesser extent knockdown of any one alone,
reduced the density of glutamatergic and GABAergic
inputs identified by immunofluorescence for the glutamate
transporter VGlut1 and the GABA transporter VGAT.
Functionally, there was a strong reduction in frequency
and amplitude of mIPSC-like events but little reduction in
amplitude of mEPSC-like events.
Regulation by alternative splicing
Neurexins undergo extensive alternate splicing, generating a huge diversity of >2000 potential variants [6,41,42].
The fact that neurexin splice insert sequences and their
positions are well conserved among neurexin genes and
among species supports the idea that alternative splicing
has important functional roles. Three of the five splice
sites occur in LNS domains, and a fourth can generate
both secreted and transmembrane forms of neurexin 3
[41]. Alternative splicing of neuroligins is much less
extensive but also occurs in the key functional domain,
the AChE-homologous region. Distinct roles of different
neurexin and neuroligin splice variants in cell–cell recognition, synaptic organization and synaptic signaling have
been suggested. Testing some of these ideas will require
Current Opinion in Neurobiology 2007, 17:43–52
generation and analysis of targeted mutations in vivo.
Nonetheless, important insights have come from three
recent studies [9,43,44] that focused on how alternative splicing in neurexins and neuroligins alters binding
affinity, hemisynapse formation and neuronal function in
culture.
These recent findings indicate that splicing at site 4 in
b-neurexins and at site B in neuroligin 1 regulate binding
selectivity and synapse function. Presence of the 30residue insert at site 4 in b-neurexin reduces the affinity
of interaction specifically with neuroligin 1 that contains
an insert at site B (+B), while maintaining high-affinity
interaction with neuroligin 1 that lacks an insert at site B
( B) or with neuroligin 2 (which lacks any B splice site
and exists only in the insert-less form) [9,43,44]. The
long a-neurexins (with or without an insert at site 4) also
bind selectively to B neuroligins [43,44]. Interestingly, it is not the nine amino acid B insert itself but
N-linked glycosylation of this insert that regulates the
interaction of neuroligin 1 with specific neurexins
[15,43,44]. Furthermore, +B neuroligin 1, which is
more selective than the B variant, comprises the
majority of neuroligin 1 in adult rat hippocampus, cortex
and cerebellum [44]. In chick sympathetic neurons, the
ratio of neurexin transcripts containing versus lacking the
insert at site 4 (+S4 versus S4) changes during development and in response to addition of growth factors in
culture [45]. Further studies are needed to determine
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Neurexin–neuroligin signaling in synapse development Craig and Kang 47
Table 1
Selectivity of SS4 b-neurexins and +B neuroligin for glutamatergic synapses, and +S4 b-neurexins and SB neuroligins for GABAergic
synapses.
A. Effect of splicing on relative binding affinities a
–S4 β-Neurexin
+S4 β-Neurexin
+B Neuroligin
–B Neuroligin
B. Induction of postsynaptic protein clustering by +S4 neurexin 1b in co-culture assays b
Proteins:
Neuroligin 1/3/4
Neuroligin 2
Clustering (%):
65 c
90 c
PSD-95
50c, 7 d
C. Enhancement of selective presynaptic input onto neurons by overexpression of neuroligin variants e
Neuroligin 1 +B
Neuroligin 1 B
Enhancement of VGlut1:
4.7d, 1.5 f
2.5 d
Enhancement of VGAT:
1.5d, 2.2 f
5.0 d
Gephyrin
104c, 110 d
Neuroligin 2 (always
5.3d, 1.5 f
5.6d, 3.0 f
B)
a
Line widths reflect affinity of interaction. b-Neurexins that contain an insert at site 4 (+S4) interact preferentially with neuroligins that lack an insert at
the B site ( B); they have lower affinity for +B neuroligin. b-Neurexins that lack an insert at site 4 ( S4) interact with B neuroligins and +B neuroligin
with equal affinity [9,43,44].
b
Induction is reported normalized to 100% induction by neurexin 1b ( S4).
c
From [9].
d
From [44].
e
Enhancement is reported as increased density of inputs immunoreactive for VGlut (a marker of glutamatergic presynapses) or VGAT (a marker of
GABAergic presynapses) relative to a value of 1.0 for control input density onto neurons expressing enhanced green fluorescent protein (EGFP). The
neuroligin forms tested all contained the A-site insert.
f
From [35].
how splicing at these key sites in neuroligin 1 (at site B)
and b-neurexins (at site 4) is regulated in specific neuron
types, during development and perhaps by activity.
These specific splice alterations of neurexins and neuroligins contribute to differences in function at GABAergic
versus glutamatergic synapses (Table 1). In co-culture
assays, addition of the site 4 insert to b-neurexin reduces
its ability to cluster the glutamate postsynaptic proteins
neuroligin 1/3/4 and PSD-95, but not the GABA postsynaptic proteins neuroligin 2 and gephyrin [9,44]. Consistent with this finding, and with the low affinity of +S4 bneurexins for +B neuroligin, addition of the B splice insert
to neuroligin 1, or artificially to neuroligin 2, reduces their
ability to cluster VGAT but not VGlut1 when overexpressed in neurons [44]. Also consistent with this idea,
neuroligin 2 (which is always B) promotes VGAT clustering more than +B neuroligin 1 [5,35]. Thus, +S4 bneurexins and
B neuroligins together selectively
promote differentiation of GABAergic synapses, whereas
S4 b-neurexins and +B neuroligin 1 together selectively
promote differentiation of glutamatergic synapses.
A few issues raised in these recent studies seem more
controversial, including the extent to which splicing
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regulates localization of neuroligins. Chih et al. [44]
reported that recombinant +B neuroligin 1 is localized
preferentially at glutamatergic synapses, whereas B neuroligin 1 is localized equally at glutamatergic and GABAergic synapses. By contrast, Graf et al. [3] reported that
artificial addition of the B splice insert to neuroligin 2 did
not alter exclusive localization of this protein to GABAergic
synapses. Furthermore, the role of alternative splicing at
the A site of neuroligins is not yet understood. The presence or absence of an insert at this site does not seem to
affect binding neuroligins to neurexins [43]. However,
Chih et al. [44] reported that addition of the A insert in the
absence of a B insert promotes localization of neuroligins to
GABAergic synapses, and that addition of the A insert to
neuroligin 1 but not to neuroligin 2 reduces ability of the
neuroligin to cluster VGlut1 but not VGAT. Thus, the A
insert might promote neuroligin localization and function
at GABAergic synapses by unknown mechanisms.
The binding of a-neurexins ( S4 or +S4) to B neuroligins
raises the question of how the roles of a-neurexins and
b-neurexins overlap in synaptic development. In
co-culture experiments, S4 neurexin 1a selectively promoted clustering of GABA postsynaptic proteins,
suggesting potential overlap in function with +S4 neurexin
Current Opinion in Neurobiology 2007, 17:43–52
48 Development
1b [44]. The phenotype of mice that lack all three aneurexins but continue to express b-neurexins (discussed
further in the following section) indicates that a-neurexins
have a unique function that is not provided by b-neurexins.
Whether b-neurexins supply a unique function not provided by a-neurexins is not yet known, but is a possibility
based on differential binding of the two neurexin forms to
+B neuroligin 1. The protein interaction studies of Boucard
et al. [43] strongly suggest that only LNS6 of a-neurexins
mediates binding to B neuroligin 1. However, the presence of alternative splice sites in the equivalent binding
surface of LNS2 and LNS4 of a-neurexins leads to speculation that these domains might also mediate regulated
binding to other partners. Indeed, only LNS2 and LNS6 of
a-neurexins that lack splice inserts bind to dystroglycan
[19]. By reducing affinity of a-neurexins for Ca2+, the
LNS2 splice inserts [10] might be expected to reduce
binding to multiple ligands. Consistent with this idea,
neurexophilins bind a-neurexin LNS2 in a Ca2+-independent manner and bind all splice variants [23]. Identification
and functional characterization of additional a-neurexin
binding partners, and determination of the role of the
secreted forms of neurexin 3 that are generated by alternative splicing at site 5, are two avenues that are ripe for
exploration.
In vivo function
The ultimate test for the functional significance of neurexins and neuroligins is to establish whether these proteins
are necessary for normal nervous system function in vivo.
This is a daunting task in mammals, but it has been
addressed heroically by generation and analysis of
Nrxn1;Nrxn2;Nrxn3 triple mutant mice that lack all three
a-neurexins [46] and of Nlgn1;Nlgn2;Nlgn3 triple neuroligin
knockout mice [47]. Interestingly, knockout of the three
a-neurexins, leaving the three b-neurexins intact, leads to
perinatal lethality due to loss of presynaptic Ca2+ channel
function [46]. Transmitter release that depends on N-type
and P/Q-type Ca2+ channels is severely reduced in these
mutants [48]. A hypothesis to reconcile these data with
those from cell culture studies of neurexins and neuroligins
is that all neurexins function as synaptic organizing molecules. a-Neurexins, via their unique extracellular
domains, are required for function and perhaps localization
of presynaptic Ca2+channels, whereas both a-neurexins
and b-neurexins contribute to presynaptic and postsynaptic localization and function of other synaptic components. We look forward to future studies of bneurexin knockout mice and complete neurexin knockout
mice, and eventually animal models altered only in neurexin and neuroligin splice composition.
Although individual neuroligin knockout mice survive
and are fertile, the Nlgn1;Nlgn2;Nlgn3 triple knockout
mice die shortly after birth owing to respiratory failure
[47]. Synapses seemed to be morphologically normal in
these mice, but there were marked functional defects in
Current Opinion in Neurobiology 2007, 17:43–52
synaptic transmission. In neurons of the pre-Bötzinger
complex, the frequency of spontaneous GABAergic/glycinergic currents was reduced by approximately 90%, and
frequency of spontaneous glutamatergic currents reduced
by approximately 75%. In Nlgn1;Nlgn2;Nlgn3 knockout
mice, the failure rate of evoked transmission was more
than tenfold greater than normal at GABAergic/glycinergic synapses, but unchanged at glutamatergic synapses.
Reduced postsynaptic clustering of GABAA receptors
seemed to be one causative factor, although no changes
in clustering of the scaffolding proteins gephyrin or PSD95 were observed. Reduced levels of several synaptic
vesicle proteins and a small reduction in the ratio of
VGAT to VGlut clusters indicate that presynaptic defects
are a contributing factor. Surprisingly, normal transmission at glutamatergic synapses in cultured neocortical
neurons indicates either a region-specific defect or one
that is compensated for in culture but not in vivo. These
data support the idea that neuroligins are essential for
recruitment of key synaptic components or for maintenance of their function. More detailed analyses of the
neuroligin knockout mice might provide further important information, especially considering their potential as
animal models of autism spectrum disorders [12].
A general question raised by the cell culture and in vivo
studies concerns the stage of synapse development at
which neurexins and neuroligins function. The co-culture
assays indicate that neurexins and neuroligins presented
locally at high concentration are sufficient to trigger
postsynaptic and presynaptic differentiation. However,
given the complex presynaptic and postsynaptic protein
networks, it might be that several molecules that bind
transmembrane components of such networks can trigger
clustering in the co-culture assays. Such molecules might
function endogenously as initial triggers of synaptogenesis, but could instead function after the initial membrane
adhesion to recruit synaptic components, and/or even
later in the process to stabilize synaptic complexes.
The interaction of soluble neuroligin 1 with neurexin
1b is of relatively low affinity (Kd 300 nM [15]) compared with the affinity of other partners such as soluble
Eph receptors for ephrins (Kd <3 nM [49]). We suggest
that other adhesion molecules such as immunoglobulindomain and cadherin family proteins mediate the initial
contact between appropriate axons and dendrites, and then
neurexins and neuroligins reinforce the contact but mainly
function to recruit and stabilize presynaptic and postsynaptic proteins. Indeed, the presence of normal numbers of
morphological synapses combined with functional defects
in the brainstem of newborn Nlgn1;Nlgn2;Nlgn3 knockout
mice strongly supports the idea that neuroligins function in
the later stages of protein recruitment or stabilization
[47]. Determining precisely when endogenous neurexins
and neuroligins cluster relative to other steps of synaptogenesis is a difficult task. By expression of tagged recombinant proteins, it has been shown that synaptic vesicle
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Neurexin–neuroligin signaling in synapse development Craig and Kang 49
clusters in axons can precede apposing clusters of PSD-95
[50], and that PSD-95–neuroligin clusters on dendrites can
precede apposing synaptic vesicle clusters [51]. A logical
prediction is that clustering of neurexins on the presynaptic
membrane and neuroligins on the postsynaptic membrane
occur simultaneously, because they would be expected to
stabilize each other [52]. This might be followed by
stabilization of synaptic vesicles by the neurexin clusters
and stabilization of postsynaptic scaffolds and receptors by
the neuroligin clusters. Neuroligins also function in
ongoing synapse maintenance, at least in cell culture
studies [3,4,5].
A second general question brought to the forefront
recently concerns what roles neurexins and neuroligins
have in development of glutamatergic versus GABAergic
synapses. The link between a-neurexins and presynaptic
Ca2+ channels is essential for function of both glutamatergic and GABAergic synapses in vivo [46]. This functional link of a-neurexins to presynaptic Ca2+ channels is
not mimicked by b-neurexins, nor does it seem to involve
neuroligins. By contrast, the neurexin–neuroligin link was
initially proposed to be specific for glutamatergic
synapses on the basis of observed protein interactions
[29]. However, results from co-culture of neurons with
neurexin-expressing cells [3], neuroligin knockdown in
culture [5] and initial neuroligin knockout studies in vivo
[47] seem to indicate stronger roles for neurexin–neuroligin interactions in function of GABAergic synapses.
Complementary studies on SynCAMs [53], ephrins and
Eph receptors [54], synaptic adhesion-like molecules
[55,56] and netrin-G ligands [57] indicate that additional
cell adhesion molecules are specific for glutamatergic as
opposed to GABAergic synapses, and that these molecules can also link to postsynaptic receptors and/or to
the presynaptic release mechanism. Perhaps there is a
greater redundancy of transmembrane molecular organizing partners at glutamatergic than at GABAergic
synapses. A clear area for further investigation is to
determine the molecular links among neuroligins and
other key postsynaptic elements of GABAergic synapses,
including GABAA receptors and gephyrin. The most
recent set of studies on alternative splicing suggest that
different splice variants of neurexins and neuroligins
function selectively at glutamatergic versus GABAergic
synapses [9,43,44]. From these culture studies, it can
be predicted that elimination of the inserts at site 4 in
neurexins in vivo might promote development of excitatory synapses and reduce that of inhibitory ones, whereas
elimination of the B insert from neuroligin 1 in vivo might
promote development of inhibitory synapses and reduce
that of excitatory ones. Whether specific neurexins and
neuroligins contribute to the matched alignment of the
appropriate postsynaptic receptor type opposite the corresponding transmitter release site is a related open
question that might be addressed using knockout or
chimera knockin mice.
www.sciencedirect.com
Clinical implications
In addition to the obvious link between neurological
disorders and the molecules that are fundamental to
synapse function, clinical interest in neuroligins was
stimulated by a report from Jamain et al. in 2003 [12].
These authors found mutations in the X-linked genes
NLGN3 and NLGN4 in siblings who had autism spectrum
disorders. The mutation in NLGN4, a frameshift that
resulted in a premature termination (396X), occurred de
novo in the mother of the affected siblings, thus pointing
strongly to a causative role. The mutation in NLGN3
resulted in a single amino acid change (R451C) within
the key AChE-homologous domain. Subsequent cell
culture studies showed that these two mutations enhance
intracellular retention of neuroligins and thus abolish or
reduce function in synaptogenesis assays [58–60].
These exciting initial findings were followed by two
confirmatory studies. In one study, another mutation in
NLGN4 that results in a premature stop (429X) was found
in all patients that were afflicted with mental retardation
with or without autism in a single family, but not in nonaffected family members or control subjects [61]. In the
second study, three missense mutations in the AChEhomologous domain (G99S, K378R and V403M) and one
in the cytoplasmic domain (R704C) were found in NLGN4
in autistic patients in a study of 148 unrelated autistic
individuals [62]. However, a limited prevalence of
mutations in NLGN3 or NLGN4 in autism is indicated
by additional studies that found no mutations in the
coding regions of these genes in a total of 416 additional
autistic patients [63–65]. Nonetheless, the association of
neuroligin mutations with a subset of autism spectrum
disorders and mental retardation opens up a molecular
and cellular avenue for further understanding and perhaps treatment of these disorders (see also review by
Geschwind and Levitt, in this issue). Specific links
between neurexins and neurological disorders have not
been reported, although the unusually large size of
NRXN1 and NRXN3 (1.1 Mb and 1.7 Mb, respectively
[42]) places them as likely candidates.
Conclusions
Accumulating studies are leading to the realization that
there might not be any single molecule, or molecular
family, that is essential for assembly of CNS synapses.
Nonetheless, neurexins and neuroligins are the best
candidates for central organizing molecules to stabilize
networks of presynaptic and postsynaptic proteins across
the synaptic cleft. Recent protein interaction assays and
cell culture experiments indicate selective functions of
splice variants: +S4 b-neurexins and B neuroligins at
GABAergic synapses versus S4 b-neurexins and +B
neuroligins at glutamatergic synapses. Different affinities
of the interactions between different neurexin–neuroligin
pairs are combined with a selective linkage to components of glutamatergic versus GABAergic synapses,
Current Opinion in Neurobiology 2007, 17:43–52
50 Development
by mechanisms that are not yet understood. However, the
significance of such a shared system is that alterations in
stoichiometry of key players can be used to regulate the
balance of excitatory and inhibitory inputs onto a neuron
[40]. Recent in vivo studies indicate that neurexins and
neuroligins have essential roles in synapse development
— not in initial adhesion, but in recruitment of molecular
components and maturation [47]. Further studies are
needed to explore the functional significance of the rich
diversity of neurexin and neuroligin variants; particularly
useful will be targeted in vivo mutagenesis and analysis of
the structure, molecular composition, function and longterm stability of glutamatergic and GABAergic synapses
in defined circuits.
Acknowledgements
We thank Ethan Graf and members of the Craig laboratory for helpful
discussions. Work in our laboratory is supported by the National Institutes
of Health (NIH) grant MH070860, Canadian Institutes for Health Research
(CIHR) grant MOP69096, Michael Smith Foundation for Health Research,
and Canada Research Chair Program.
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